Abstract: This project is aimed at the study of properties of membrane proteins that underlie important biomedical processes and are implicated in health disorders and how they interact with their membrane environments. By means of the latest one and two dimensional electron-spin resonance (ESR) methodologies, including Pulse-Dipolar ESR (PDS), multifrequency ESR, and Two-Dimensional Electron-Electron Double Resonances (2D-ELDOR), as well as site-directed nitroxide spin-labeling methods, we will directly observe significant protein functional changes and in parallel experiments, observe the concomitant changes occurring in the dynamic structure of the lipid bilayers. This ability to accurately characterize both membrane and membrane protein is an innovative approach toward understanding on the role of membrane-protein interactions affecting the protein's function. Specific projects include the following. First, we will advance our study of the mechanism of viral membrane fusion, based on our previous success in detecting the membrane ordering effect of influenza and HIV glycoprotein fusion peptides (FP). We will extend this study to the structure of their FP-transmembrane domain complex in membranes and to quantification of the induced microdomain, plus we shall extend the study to SARS and gamete membrane fusogens. In the second project, the tau protein, which plays an important role in neurodegeneration, will be studied. We have shown how tau interacts with membranes and adopts different conformations in response to the curvature of liposomes, so we plan to study the interaction between tau and microtubules, which directly addresses its functional and pathological roles in neurodegenerative diseases. The third project will focus on the structure of asymmetric membranes, which are crucial for cell function. We will investigate how both membrane leaflets interact with each other, and how the changes in the composition of one leaflet affect ordering and fluidity of the other, as well as how the model peptide gramicidin changes their structure and facilitates lipid flip-flop. In the fourth project, we will study the influenza A M1 and M2 matrix proteins, which are related to viral infectivity and proliferation. We previously showed the oligomerization of M2 TMD in membrane is a two-step process and the stoichiometry is affected by ligand binding. We will focus on the effect of lipid composition on the M1 oligomerization. We will also study the structure of the M1-M2 complex in membranes. In the fifth project, our ESR studies on the mechanism of transmembrane signaling in bacterial chemoreceptors will be continued and extended. We will 1) characterize the lipid dependence of the piston motion exhibited by chemoreceptors, 2) examine the effect of receptor oligomerization state and conformation on lipid structure, and 3) probe the interactions of the chemoreceptor sensing domain relative to the lipid bilayer. All these studies will involve extensive collaborations with leading research groups. Possible clinical applications include detection of membrane changes during immune response, prevention of viral entry, fertility diseases, neurological disorders, and development of antimicrobials.